PROJECT SUMMARY/ABSTRACT The APOBEC3 family (APOBEC3A-H) of single-stranded DNA cytidine deaminases consists of seven proteins (APOBEC3A-H). These proteins act as restriction factors against a variety of viruses including HIV. Four of these proteins, APOBEC3D-H, are known to restrict HIV by being packaged into virions and mutating ssDNA during reverse transcription during the next round of infection. APOBEC3A (A3A) stands out against the other APOBEC3 family members because of its unique, highly restricted expression: it is expressed only in myeloid cells (macrophages, monocytes, and dendritic cells) in response to interferon stimulation. Moreover, unlike the other APOBEC3 proteins, A3A is not packaged into virions and has been shown to block the early steps of HIV replication in interferon stimulated primary monocytes and macrophages pointing to a novel, non-canonical mechanism of restriction. APOBEC3A also does not interact with Vif, an HIV accessory protein which confers resistance to other APOBEC3 proteins by marking them for degradation in the proteasome. Little is currently known about how A3A targets HIV and how its expression is regulated in such an unusual manner. We propose two Specific Aims to elucidate the mechanism by which A3A targets HIV and how its expression is controlled at the molecular level. In Specific Aim 1, we will dissect the role A3A plays in HIV infection. In preliminary studies, we have used the CRISPR/Cas9-based synergistic activation mediator system to specifically activate endogenous A3A transcription in human T cells, which generally fail to express this intracellular inhibitor. We will use these engineered T cell lines to test the degree to which and the mode of action by which A3A targets HIV infection. Using this same system, we will also examine if A3A expression alone is sufficient to render primary T cells resistant to HIV. We will also conduct knockout experiments in primary human monocytes using electroporation-mediated delivery of CRISPR ribonucleoprotein (crRNP). This allows for efficient knockout of genes in these cells. In Specific Aim 2, we propose to examine the factors controlling the unique expression pattern of A3A. Using publicly available large datasets, we identify regions upstream of the A3A transcription start site that are unique to A3A and are absent upstream of any other APOBEC3 coding region. We will clone these regions of interest and use different approaches including luciferase assays to dissect how these regions influence A3A transcription. Variation in regulation of A3A may influence susceptibility to HIV as well as represent a risk factor for a variety of human malignancies. Taken together, our findings will shape our understanding of the mode of action, the regulation and the biological function of this powerful intra-cellular DNA mutator protein at the interface of antiviral immunity and cancer.